RNA and DNA cloning techniques, in which RNA or DNA fragments are inserted into suitable cloning vectors, are widely used for identifying specific cloned sequences and for expression of the cloned fragments. In a typical cloning method, total RNA is extracted from a selected source, such as a body fluid, cell, or tissue source, and a poly A RNA fraction, representing predominantly messenger RNA (mRNA), is copied to produce double-strand cDNA fragments. The cDNA fragments are then ligated with restriction site linkers, to provide restriction-site ends for cloning into a selected site in a suitable cloning vector. A fragment library formed from the fragments can be used for hybridization to specific probes, and/or for production of specific coded-for proteins or peptides.
Although the above method has yielded a number of impressive successes, it nonetheless has several limitations. First, where the amount of source material is quite small, e.g., 10.sup.3 -10.sup.4 cells, the total amount of extracted RNA and resultant cDNA may be too small to produce a library with an adequate, i.e., representative, number of cloned sequences. Secondly, since the duplex fragments have the same linkers at each end, directional cloning in the cloning vector is precluded. Finally, a large percentage of the RNA species are only partially copied, so that many of the library clones are lacking 5'-end coding regions.
There are other biochemical genetics techniques which are also limited by the amount of available nucleic acid material. For example, it is often of interest to compare the RNA species produced by two related sources, to attempt to identify species which are unique to one source or the other. Since the unique species may exist as only a very small fraction of the total material, a relatively large amount of starting material is needed to isolate the unique sequence(s) of interest.
Similarly, where the source material is suspected of containing a disease agent of interest, at very low concentration, it may be impossible to detect the presence of the sequence of interest by conventional cloning or probe hybridization methods.
Recently, a method for amplifying duplex DNA fragments by repeated strand-replication has been described (U.S. Pat. Nos. 4,683,194 and 4,683,202). This method, generally referred to as a polymerase chain reaction (PCR) method, is designed to selectively amplify fragments containing different known sequences in the two fragment strands, using two primers which are homologous to the two different-sequence regions. The PCR method previously described is of course suitable for amplifying specific fragments for which two different sequence-specific probes exist. However, the described method is not intended for or applicable to the problem of non-specifically amplifying fragments in a fragment mixture containing different sequences.